The present invention relates to a spray-dried bacteriocin powder with anti-microbial activity, and to a method of producing the powder. In particular, the invention relates to a lacticin 3147 spray-dried powder.
1. Prior Art
The elimination of food spoilage and pathogenic organisms has become the focus of much research since, in terms of individuals affected and the cost of treatment, food-borne illnesses have an enormous impact. It has been estimated that microbial pathogens in food cause 6.5-33 million cases of human illness annually in the U.S., at a cost of between $2.9-$6.7 billion dollars (2), with Gram-positive food-borne pathogens accounting for between 25-55% of the costs. In recent years, consumer demand for fresh minimally processed safe food, in addition to concern over the use of chemical preservatives in foods, has prompted substantial interest in the application of biopreservatives. Bacteriocins produced by lactic acid bacteria are seen as alternatives to traditional preservatives for ensuring food safety and potential applications in foods have been readily identified (21).
Nisin, a bacteriocin produced by certain strains of Lactococcus lactis, has been used successfully to control food spoilage, in a number of different foods, including cheeses, canned goods and dairy desserts (10). However, its use is subject to certain restrictions. It is most effective in foods with acidic pH (below pH 6.0) and low protein and fat content. It is poorly soluble above pH 6.0 and as such has limited effectiveness in many foods. A powdered form of nisin, Nisaplin (Aplin and Barrett, Towbridge, Wiltshire, U.K.) has been developed and is used for the preservation of foods.
In addition to the development of Nisaplin, other powdered bacteriocin-containing agents have been developed for the preservation of foods. Propionibacterium freundenreichii subsp. shermanni is used to produce Microgard (Wesman Foods, Inc., Beaverton, Oreg.) by pasteurisation and drying of propionibacteria-fermented skim milk. It is estimated to be used in approximately one third of all cottage cheese made in the US and is said to be inhibitory to most Gram-negative bacteria and some fungi (4). The active agents in Microgard include propionic acid, acetic acid, diacetyl, lactic acid and a heat-stable peptide of approximately 700 daltons which is considered to be the most active component.
Lacticin 3147 is a bacteriocin produced by L. lactis DPC3147 which has a similar host range to that of nisin, in that it is inhibitory to a wide range of Gram-positive organisms, including Listeria, Clostridium spp., Enterococcus, Staphylococcus and Streptococcus (17). Given that many of these organisms have been identified as agents of food spoilage and pathogenesis, the development of a lacticin 3147-based system for control of these organisms has obvious attractions. This may be achieved in two ways. The first involves the use of starter cultures (including transconjugants) which produce lacticin 3147, and can be used in food fermentations where these strains can be substituted for the original starter cultures. The genetic determinants for lacticin 3147 are encoded on a 60.2 kb plasmid, pMRC01 which has been fully sequenced (6) and which has been mobilised to a number of cheese starter cultures (3). Lacticin 3147 is the subject of PCT Application No. PCT/IE96/00022, published as WO 96/32482.
Recently, it has been shown that a lacticin 3147 producing transconjugant can inhibit Listeria monocytogenes in Cottage cheese (13). This starter has also been used to control the proliferation of non-starter lactic acid bacteria in Cheddar cheese. The second approach to improving food safety through the use of lacticin 3147 involves the development of a spray-dried form of the bacteriocin. The advantage of such a bio-active powder is that it could be applied as a food ingredient in a variety of foods. However, it is not at all apparent that the bacteriocin is robust enough to withstand spray-drying and there was the possibility that spray-drying would result in a significant loss in bacteriocin activity.
The object of the invention is to provide a lacticin 3147-enriched food ingredient for incorporation into foodstuffs. In particular, it is an object to provide a spray-dried lacticin 3147 powder. It could not be predicted that such a spray-dried powder could be produced since spray-drying could have caused heat denaturation of the bacteriocin, bearing in mind that lacticin 3147 is composed of two peptides, both of which are required for activity. Furthermore, dehydration could irreversibly inactivate the bacteriocin.
Described herein is a whey based bio-active powder, with effectiveness in controlling two representative pathogens, L. monocytogenes and Staphylococcus aureus, in buffer at both neutral and acidic pH. Also described is its effectiveness in controlling L. monocytogenes in an infant milk formulation and other foodstuffs. However, it will be apparent to those skilled in the art that the bacteriocin-powder of the invention need not be dairy based and that it would also be possible to produce a spray-dried bacteriocin based, for example, on other powders, synthetic materials or the like.
According to the present invention there is provided a process for the production of spray-dried lacticin 3147 powder comprising:
(a) inoculating a medium with a lacticin 3147-producing strain of bacteria;
(b) fermenting the inoculated medium;
(c) adjusting the pH of the fermentation to pH 6.3 to 6.7;
(d) inactivating the bacterial fermentate;
(e) evaporating the fermentate of step (d).
The medium which may be inoculated with the bacteria can be selected from milk or dairy-based powders including demineralised whey powder, reconstituted skimmed milk powder, whey protein concentrate powder, pasteurised milk, Cheddar cheese whey, or synthetic laboratory media such as LM17 or TY broth or the like.
Preferably the inoculated medium is fermented at about 30xc2x0 C. for about 6 to 24 hours.
Preferably the pH of the fermentation is adjusted to about 6.5.
Suitably, the bacterial fermentate is inactivated by pasteurisation or treating at ultra-high temperature.
Suitably, if the fermentate is pasteurised, it is pasteurised at about 72xc2x0 C. for about 15 seconds.
Preferably the inactivated fermentate is evaporated at about 60xc2x0 C. to about 40% total solids.
The concentrate of step (e) may then be cooled to about 32xc2x0 C., seeded with lactose at about 0.1% w/w and allowed to crystallise at a cooling rate of about 1xc2x0 C. per hour.
The crystallised concentrate is then spray-dried by methods known in the art.
The invention also provides a spray-dried lacticin 3147 powder which has the ability to inhibit organisms which are not resistant to lacticin 3147 and which may suitably have an activity of about 40,240 au (arbitary units)/per ml.
The invention also provides a food product comprising a spray-dried lacticin 3147 powder as defined above. The food product may be an infant milk formulation, a sauce, mayonnaise, a dessert, a yoghurt, a custard, a tinned food product such as a tinned vegetable or tinned meat product, a soup, a bakery product or similar products.
The food product may further have been subjected to increased hydrostatic pressure during processing, suitably at a pressure of about 150 to 800 MPa.